Summary. Dysregulation of PTEN and MKP-1/DUSP1 by changes in expression/degradation, mutation, and/or post-translational modifications (PTMs) contribute to the progression of human diseases such as different cancers. MKP-1 (aka DUSP1) and PTEN are major negative regulatory phosphatase enzymes in the MAPK and PI3K/AKT cell signaling pathways, and both enzymes are subject to PTMs mapped to their C- terminal tails. MKP-1 is phosphorylated twice at positions 359 and 364, whereas PTEN is tetra-phosphorylated at positions 380, 382, 383, and 385 and acetylated at position 402 but there are large gaps in our understanding of the structural and functional impacts of these PTMs. Therefore, our goal is to elucidate the molecular basis for the PTM-dependent regulation of PTEN and MKP-1. Key to our proposal is the generation of semi-synthetic enzymes by expressed protein ligation that facilitates site-specific and stoichiometric insertion of the corresponding PTM(s) and their mimics to be use in these three specific aims: Specific aim 1 will determine the structural basis for the phosphorylation-dependent conformational closure of PTEN. Direct atomic interactions for the phosphorylated C-terminal tail with the PTEN body will be mapped by UV-induced photocrosslinking, which will contribute to a computational model that defines the position of the C-terminal tail when in the closed conformational state. The computational model will be tested by generating site-directed mutants and evaluating them with a series of biochemical assays, including enzyme activity, sensitivity to dephosphorylation by alkaline phosphatase, and lipid binding. Specific aim 2 utilizes a series of biochemical and cellular techniques to define the function of C-terminal (Lys402) acetylation of PTEN. This aim concerns the role of Lys402 acetylation in modulating PTEN's biochemical and cellular function(s) by elucidating its role in regulating enzyme activity, protein-protein interactions, subcellular localization, protein stability, and regulation of different signaling pathways. A series of biochemical and cellular methods will be executed to systematically define the regulatory role that this PTM has on PTEN's cellular function. Specific aim 3 employs a systematic approach to evaluate the role C-terminal phosphorylation has on the biochemical and cellular function of MKP-1. This aim seeks to define the biochemical and/or cellular consequences for these phosphorylation events with respect to enzyme activity, protein-protein interactions, subcellular localization, protein stability, and regulation of different signaling pathways. A series of biochemical and cellular assays will be employed to define how C-terminal phosphorylation regulates MKP-1 function. We anticipate discovering unique regulatory mechanisms for these PTMs that can pave the way to a deeper understanding of cell signaling and next generation therapies.